Peptide-metal chelate conjugates

ABSTRACT

This invention relates to diagnostic and radiodiagnostic agents, including radiolabeled scintigraphic imaging agents, and therapeutic and radiotherapeutic agents. The invention provides such agents and reagents for preparing such agents, and methods for producing and using such reagents. Specifically, the invention provides radiolabeled imaging agents and non-radioactively labeled imaging agents for imaging sites in a mammalian body and reagents for preparing these imaging agents. The invention also provides radiolabeled therapeutic agents, as well as non-radioactively labeled therapeutic agents, and reagents and methods for preparing these agents. The agents and reagents provided comprise a specific binding peptide, covalently linked to a metal ion-complexing moiety. Reagents, methods and kits for making such reagents, methods for labeling such reagents, and methods for using such labeled reagents are provided.

This application is a divisional of U.S. patent application, Ser. No.08/241,625, filed May 12, 1994, now U.S. Pat. No. 5,783,170 which is acontinuation-in-part of U.S. Ser. No. 07/807,062, filed Nov. 27, 1991,now U.S. Pat. No. 5,443,815, issued Aug. 22, 1995. The disclosures ofeach of these prior applications are considered as being part of thedisclosure of the application and are explicitly incorporated byreference therein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to therapeutic agents, radiotherapeutic agents,radiodiagnostic agents, and non-radioactive diagnostic agents, andmethods for producing such diagnostic and therapeutic agents. Theinvention also relates to cyclic peptides which specifically bind tosomatostatin receptors expressed at the surface of mammalian cells,particularly neoplastic or metastatic mammalian cells. The invention inone aspect relates to scintigraphic imaging agents for imaging sites ina mammalian body. In this aspect, the imaging agents comprise aspecific-binding peptide that specifically binds to somatostatinreceptor-expressing cells in vivo, labeled with technetium-99m (Tc-99m)via a radiolabel-binding moiety which forms a complex with Tc-99m. Inanother aspect, the invention provides radioiodinated imaging agents.The invention also provides therapeutic agents, including radioiodinatedagents, radioactive metal-reagent complexes and nonradioactivemetal-reagent complexes, all of which have therapeutic utility. Theinvention provides reagents for preparing each of the diagnostic andtherapeutic embodiments of the diagnostic and therapeutic agents of theinvention, the radiolabeled embodiments and non-radioactive metalcomplexes thereof, methods for labeling said reagents and kitscomprising non-radioactive embodiments of the reagents of the inventionand other components for the convenient preparation of the radiolabeleddiagnostic and therapeutic agents of the invention.

2. Description of the Prior Art

Somatostatin is a tetradecapeptide that is endogenously produced by thehypothalamus and pancreas in humans and other mammals. The peptide hasthe formula: ##STR1## (Single letter abbreviations for amino acids canbe found in G. Zubay, Biochemistry (2d ed.), 1988, (MacMillanPublishing: New York), p.33). This peptide exerts a wide variety ofbiological effects in vivo. It is known to act physiologically on thecentral nervous system, the hypothalamus, the pancreas, and thegastrointestinal tract.

Somatostatin inhibits the release of insulin and glucagon from thepancreas, inhibits growth hormone release from the hypothalamus, andreduces gastric secretions. Thus, somatostatin has clinical andtherapeutic applications for the alleviation of a number of ailments anddiseases, both in humans and other animals. Native somatostatin is oflimited utility, however, due to its short half-life in vivo, where itis rapidly degraded by peptidases. For this reason, somatostatinanalogues having improved in vivo stability have been developed in theprior art.

Freidinger, U.S. Pat. No. 4,235,886 disclose cyclic hexapeptidesomatostatin analogues useful in the treatment of a number of diseasesin humans.

Coy and Murphy, U.S. Pat. No. 4,485,101 disclose synthetic dodecapeptidesomatostatin analogues.

Freidinger, U.S. Pat. No. 4,611,054 disclose cyclic hexapeptidesomatostatin analogues useful in the treatment of a number of diseasesin humans.

Nutt, U.S. Pat. No. 4,612,366 disclose cyclic hexapeptide somatostatinanalogues useful in the treatment of a number of diseases in humans.

Coy et al., U.S. Pat. No. 4,853,371 disclose synthetic octapeptidesomatostatin analogues.

Coy and Murphy, U.S. Pat. No. 4,871,717 disclose synthetic heptapeptidesomatostatin analogues.

Coy et al., U.S. Pat. No. 4,904,642 disclose synthetic octapeptidesomatostatin analogues.

Taylor et al., U.S. Pat. No. 5,073,541 disclose a method of treatingsmall cell lung cancer.

Brady, European Patent Application No. 83111747.8 discloses dicyclichexapeptide somatostatin analogues useful in the treatment of a numberof human diseases.

Bauer et al., European Patent Application No. 85810617.2 disclosesomatostatin derivatives useful in the treatment of a number of humandiseases.

Eck and Moreau, European Patent Application No. 90302760.5 disclosetherapeutic octapeptide somatostatin analogues.

Coy and Murphy, International Patent Application Serial No.PCT/US90/07074 disclose somatostatin analogues for therapeutic uses.

Schally et al., European Patent Application Serial No. EPA 911048445.2disclose cyclic peptides for therapeutic use.

Bodgen and Moreau, International Patent Application Serial No.PCT/US92/01027 disclose compositions and methods for treatingproliferative skin disease.

Somatostatin exerts its effects by binding to specific receptorsexpressed at the cell surface of cells comprising the central nervoussystem, the hypothalamus, the pancreas, and the gastrointestinal tract.These high-affinity somatostatin binding sites have been found to beabundantly expressed at the cell surface of most endocrine-active tumorsarising from these tissues.

It is frequently clinically advantageous for a physician to be able tolocalize the site of pathological conditions in a patient usingnon-invasive means. Such pathological conditions include diseases of thelungs, heart, liver, kidneys, bones and brain, as well as cancer,thrombosis, pulmonary embolism, infection, inflammation andatherosclerosis.

In the field of nuclear medicine, certain pathological conditions arelocalized, or their extent is assessed, by detecting the distribution ofsmall quantities of internally-administered radioactively labeled tracercompounds (called radiotracers or radiopharmaceuticals). Methods fordetecting these radiopharmaceuticals are known generally as imaging orradioimaging methods.

In radioimaging, the radiolabel is a gamma-radiation emittingradionuclide and the radiotracer is located using a gamma-radiationdetecting camera (this process is often referred to as gammascintigraphy). The imaged site is detectable because the radiotracer ischosen either to localize at a pathological site (termed positivecontrast) or, alternatively, the radiotracer is chosen specifically notto localize at such pathological sites (termed negative contrast). Inmany situations it is a particular advantage to use a radiolabeledspecific binding compound as a radiopharmaceutical which localizesspecifically to the pathological site in vivo.

For example, expression of high-affinity binding sites for somatostatinis a marker for certain tumor cells, and specific binding withsomatostatin can be exploited to locate and identify such tumor cells invivo.

Methods for radiolabeling somatostatin analogues that have been modifiedso as to contain a tyrosine amino acid (Tyr or Y) are known in the priorart.

Albert et al., UK Patent Application 8927255.3 disclose radioimagingusing somatostatin derivatives such as octreotide labeled with ¹²³ I.

Bakker et al., 1990, J. Nucl. Med. 31: 1501-1509 describe radioactiveiodination of a somatostatin analog and its usefulness in detectingtumors in vivo.

Bakker et al., 1991, J. Nucl. Med. 32: 1184-1189 teach the usefulness ofradiolabeled somatostatin for radioimaging in vivo.

Bomanji et al., 1992, J. Nucl. Med. 33: 1121-1124 describe the use ofiodinated (Tyr-3) octreotide for imaging metastatic carcinoid tumors.

Alternatively, methods for radiolabeling somatostatin by covalentlymodifying the peptide to contain a radionuclide-chelating group havebeen disclosed in the prior art.

Albert et al., UK Patent Application 8927255.3 disclose radioimagingusing somatostatin derivatives such as octreotide labeled with ¹¹¹ Invia a chelating group bound to the amino-terminus.

Albert et al., International Patent Application No. WO 91/01144 discloseradioimaging using radiolabeled peptides related to growth factors,hormones, interferons and cytokines and comprised of a specificrecognition peptide covalently linked to a radionuclide chelating group.

Albert et al., European Patent Application No. 92810381.1 disclosesomatostatin peptides having amino-terminally linked chelators.

Faglia et al., 1991, J. Clin. Endocrinol. Metab. 73: 850-856 describethe detection of somatostatin receptors in patients.

Kwekkeboom et al., 1991, J. Nucl. Med. 32: 981 Abstract #305 relates toradiolabeling somatostatin analogues with ¹¹¹ In.

Albert et al., 1991, Abstract LM10, 12th American Peptide Symposium:1991 describe uses for ¹¹¹ In-labeled diethylene-triaminopentaaceticacid-derivatized somatostatin analogues.

Krenning et al., 1992, J. Nucl. Med. 33: 652-658 describe clinicalscintigraphy using (¹¹¹ In)(DTPA)octreotide.

A variety of radionuclides are known to be useful for radioimaging,including ⁶⁷ Ga, ^(99m) Tc (Tc-99m), ¹¹¹ In, ¹²³ I, ¹²⁵ I, ¹⁶⁹ Yb or ¹⁸⁶Re. A number of factors must be considered for optimal radioimaging inhumans. To maximize the efficiency of detection, a radionuclide thatemits gamma energy in the 100 to 200 keV range is preferred. To minimizethe absorbed radiation dose to the patient, the physical half-life ofthe radionuclide should be as short as the imaging procedure will allow.To allow for examinations to be performed on any day and at any time ofthe day, it is advantageous to have a source of the radionuclide alwaysavailable at the clinical site. Tc-99m is a preferred radionuclidebecause it emits gamma radiation at 140 keV, it has a physical half-lifeof 6 hours, and it is readily available on-site using amolybdenum-99/technetium-99m generator. Other radionuclides used in theprior art are less advantageous than Tc-99m. This can be because thephysical half-life of such radionuclides are longer, resulting in agreater amount of absorbed radiation dose to the patient (e.g.,indium-111). Also, many disadvantageous radionuclides cannot be producedusing an on-site generator.

Tc-99m is a transition metal that is advantageously chelated by a metalcomplexing moiety. Radiolabel complexing moieties capable of bindingTc-99m can be covalently linked to various specific binding compounds toprovide a means for radiolabeling such specific binding compounds. Thisis because the most commonly available chemical species of Tc-99m,pertechnetate (TcO₄ ⁻), cannot bind directly to most specific bindingcompounds strongly enough to be useful as a radiopharmaceutical.Complexing of Tc-99m with such radiolabel complexing moieties typicallyentails chemical reduction of the pertechnetate using a reducing agentsuch as stannous chloride.

The use of chelating agents for complexing Tc-99m is known in the priorart.

Byrne et al., U.S. Pat. No. 4,434,151 describe homocysteine containingchelating agents for Tc-99m.

Fritzberg, U.S. Pat. No. 4,444,690 describes a series oftechnetium-chelating agents based on2,3-bis(mercaptoacetamido)propanoate.

Byrne et al., U.S. Pat. No. 4,571,430 describe homocysteine containingchelating agents for Tc-99m.

Byrne et al., U.S. Pat. No. 4,575,556 describe homocysteine containingchelating agents for Tc-99m.

Nosco et al., U.S. Pat. No. 4,925,650 describe Tc-99m chelatingcomplexes.

Kondo et al., European Patent Application, Publication No. 483704 A1disclose a process for preparing a Tc-99m complex with amercapto-Gly-Gly-Gly moiety.

European Patent Application No. 84109831.2 describes bisamido, bisthiolTc-99m ligands and salts thereof as renal function monitoring agents.

Davison et al., 1981, Inorg. Chem. 20: 1629-1632 disclose oxotechnetiumchelate complexes.

Fritzberg et al., 1982, J. Nucl. Med. 23: 592-598 disclose a Tc-99mchelating agent based on N,N'-bis(mercaptoacetyl)-2,3-diaminopropanoate.

Byrne et al., 1983, J. Nucl. Med. 24: P126 describe homocysteinecontaining chelating agents for Tc-99m.

Bryson et al., 1988, Inorg. Chem. 27: 2154-2161 describe neutralcomplexes of technetium-99 which are unstable to excess ligand.

Misra et al., 1989, Tet. Lett. 30: 1885-1888 describe bisamine bisthiolcompounds for radiolabeling purposes.

The use of chelating agents for radiolabeling specific-binding compoundsis known in the art.

Gansow et al., U.S. Pat. No. 4,472,509 teach methods of manufacturingand purifying Tc-99m chelate-conjugated monoclonal antibodies.

Stavrianopoulos, U.S. Pat. No. 4,943,523 teach detectable moleculescomprising metal chelating moieties.

Fritzberg et al., European Patent Application No. 86100360.6 describedithiol, diamino, or diamidocarboxylic acid or amine complexes usefulfor making technetium-labeled imaging agents.

Albert et al., UK Patent Application 8927255.3 disclose radioimagingusing somatostatin derivatives such as octreotide labeled with ¹¹¹ Invia a chelating group bound to the amino-terminus.

Albert et al., International Patent Application, Publication No. WO91/01144 disclose radioimaging using radiolabeled peptides related togrowth factors, hormones, interferons and cytokines and comprised of aspecific recognition peptide covalently linked to a radionuclidechelating group.

Fischman et al., International Patent Application, Publication No.WO93/13317 disclose chemotactic peptides attached to chelating moieties.

Kwekkeboom et al., 1991, J. Nucl. Med. 32: 981 Abstract #305 relates toradiolabeling somatostatin analogues with ¹¹¹ In.

Albert et al., 1991, Abstract LM10, 12th American Peptide Symposium:1991 describe uses for ¹¹¹ In-labeled diethylene-triaminopentaaceticacid-derivatized somatostatin analogues.

Cox et al., 1991, Abstract, 7th International Symposium onRadiopharmacology, p. 16, disclose the use of, Tc-99m-, ¹³¹ I- and ¹¹¹In-labeled somatostatin analogues in radiolocalization of endocrinetumors in vivo by scintigraphy.

Methods for labeling certain specific-binding compounds with Tc-99m areknown in the prior art.

Hnatowich, U.S. Pat. No. 4,668,503 describe Tc-99m proteinradiolabeling.

Tolman, U.S. Pat. No. 4,732,684 describe conjugation of targetingmolecules and fragments of metallothionein.

Nicolotti et al., U.S. Pat. No. 4,861,869 describe bifunctional couplingagents useful in forming conjugates with biological molecules such asantibodies.

Fritzberg et al., U.S. Pat. No. 4,965,392 describe various S-protectedmercaptoacetylglycylglycine-based chelators for labeling proteins.

Schochat et al., U.S. Pat. No. 5,061,641 disclose direct radiolabelingof proteins comprised of at least one "pendent" sulfhydryl group.

Fritzberg et al., U.S. Pat. No. 5,091,514 describe various S-protectedmercaptoacetylglycylglycine-based chelators for labeling proteins.

Gustavson et al., U.S. Pat. No. 5,112,953 disclose Tc-99m chelatingagents for radiolabeling proteins.

Kasina et al., U.S. Pat. No. 5,175,257 describe various combinations oftargeting molecules and Tc-99m chelating groups.

Dean et al., U.S. Pat. No. 5,180,816 disclose methods for radiolabelinga protein with Tc-99m via a bifunctional chelating agent.

Sundrehagen, International Patent Application, Publication No.WO85/03231 disclose Tc-99m labeling of proteins.

Reno and Bottino, European Patent Application 87300426.1 discloseradiolabeling antibodies with Tc-99m.

Bremer et al., European Patent Application No. 87118142.6 discloseTc-99m radiolabeling of antibody molecules.

Pak et al., International Patent Application, Publication No. WO88/07382 disclose a method for labeling antibodies with Tc-99m.

Goedemans et al., International Patent Application, Publication No. WO89/07456 describe radiolabeling proteins using cyclic thiol compounds,particularly 2-iminothiolane and derivatives.

Dean et al., International Patent Application, Publication No.WO89/12625 teach bifunctional coupling agents for Tc-99m labeling ofproteins.

Schoemaker et al., International Patent Application, Publication No.WO90/06323 disclose chimeric proteins comprising a metal-binding region.

Thornback et al., EPC Application No. 90402206.8 describe preparationand use of radiolabeled proteins or peptides using thiol-containingcompounds, particularly 2-iminothiolane.

Gustavson et al., International Patent Application, Publication No.WO91/09876 disclose Tc-99m chelating agents for radiolabeling proteins.

Rhodes, 1974, Sem. Nucl. Med. 4: 281-293 teach the labeling of humanserum albumin with technetium-99m.

Khaw et al., 1982, J. Nucl. Med. 23: 1011-1019 disclose methods forlabeling biologically active macromolecules with Tc-99m.

Schwartz et al., 1991, Bioconjugate Chem. 2: 333 describe a method forlabeling proteins with Tc-99m using a hydrazinonicotinamide group.

Attempts at radiolabeling peptides have been reported in the prior art.

Ege et al., U.S. Pat. No. 4,832,940 teach radiolabeled peptides forimaging localized T-lymphocytes.

Morgan et al., U.S. Pat. No. 4,986,979 disclose methods for imagingsites of inflammation.

Flanagan et al., U.S. Pat. No. 5,248,764 describe conjugates between aradiolabel chelating moiety and atrial natiuretic factor-derivedpeptides.

Ranby et al., 1988, PCT/US88/02276 disclose a method for detectingfibrin deposits in an animal comprising covalently binding aradiolabeled compound to fibrin.

Lees et al., 1989, PCT/US89/01854 teach radiolabeled peptides forarterial imaging.

Morgan et al., International Patent Application, Publication No.WO90/10463 disclose methods for imaging sites of inflammation.

Flanagan et al., European Patent Application No. 90306428.5 discloseTc-99m labeling of synthetic peptide fragments via a set of organicchelating molecules.

Stuttle, PCT Application, Publication No. WO 90/15818 describes Tc-99mlabeling of RGD-containing oligopeptides.

Rodwell et al., 1991, PCT/US91/03116 disclose conjugates of "molecularrecognition units" with "effector domains".

Cox, International Patent Application No. PCT/US92/04559 disclosesradiolabeled somatostatin derivatives containing two cysteine residues.

Rhodes et al., International Patent Application, Publication No.WO93/12819 teach peptides comprising metal ion-binding domains.

Lyle et al, International Patent Application, Publication No. WO93/15770disclose Tc-99m chelators and peptides labeled with Tc-99m.

Coughlin et al, International Patent Application, Publication No.WO93/21151 disclose bifunctional chelating agents comprising thioureagroups for radiolabeling targeting molecules.

Knight et al., 1990, 37th Annual Meeting of the Society of NuclearMedicine, Abstract #209, claim thrombus imaging using Tc-99m labeledpeptides.

Babich et al., 1993, J. Nucl. Med. 34: 1964-1974 describe Tc-99m labeledpeptides comprising hydrazinonicotinamide derivatives.

Methods for directly labeling somatostatin, derivatives of somatostatin,analogues of somatostatin or peptides that bind to the somatostatinreceptor and contain at least 2 cysteine residues that form a disulfideor wherein the disulfide is reduced to the sulfhydryl form, aredisclosed in co-owned U.S. Pat. No. 5,225,180, issued Jul. 6, 1993 whichis hereby incorporated by reference in its entirety.

The use of chelating agents for radiolabeling peptides, and methods forlabeling peptides with Tc-99m are known in the prior art and aredisclosed in co-pending U.S. patent applications Ser. No. 07/653,012,now abandoned, which issued as U.S. Pat. No. 5,811,394; Ser. No.07/807,062, now U.S. Pat. No. 5,443,815; Ser. Nos. 07/871,282;07/893,981, now U.S. Pat. No. 5,508,020; Ser. No. 07,/955,466, nowabandoned; Ser. Nos. 08/092,355; and 08/095,760, now U.S. Pat. No.5,620,675.

There remains a need for synthetic (to make routine manufacturepracticable and to ease regulatory acceptance) somatostatin analogueshaving increased in vivo stability, to be used therapeutically, asscintigraphic agents when radiolabeled with Tc-99m or other detectableradioisotopes for use in imaging tumors in vivo, and as radiotherapeuticagents when radiolabeled with a cytotoxic radioisotope such asrhenium-188. Small synthetic somatostatin analogues are provided by thisinvention that specifically fulfill this need.

SUMMARY OF THE INVENTION

The present invention provides somatostatin analogues that comprisecyclic peptides for therapeutic applications, including radiotherapeuticapplications, and diagnostic applications, including radiodiagnosticapplications, in particular scintigraphic imaging applications. Distinctfrom native somatostatin, the cyclic peptides of the invention are notcomprised of a disulfide bond. The invention also provides cyclicpeptide reagents comprised of cyclic peptide somatostatin analogueswherein such peptides incorporate a covalently linked metal ion-bindingmoiety. The invention provides such cyclic peptides, cyclic peptidereagents and radiolabeled cyclic peptide reagents that are scintigraphicimaging agents, radiodiagnostic agents and radiotherapeutic agents.Scintigraphic imaging agents of the invention comprise cyclic peptidereagents radiolabeled with a radioisotope, preferably technetium-99m.Radiotherapeutic agents of the invention comprise cyclic peptidereagents radiolabeled with a cytotoxic radioisotope, preferablyrhenium-186 or rhenium-188. Methods for making and using such cyclicpeptides, cyclic peptide reagents and radiolabeled embodiments thereofare also provided.

The invention provides compositions of matter comprising a specificbinding peptide that specifically binds to somatostatin receptors in amammalian body, covalently linked to a metal ion-complexing moiety. Thecompositions of matter of the invention have formula:

    cyclo(N--CH.sub.3)-Phe-Tyr-(D-Trp)-Lys-Val-Hcy.(CH.sub.2 CO.X)

wherein X is (amino acid)_(n) -B-(amino acid)_(m) -Z, wherein B is athiol-containing moiety that is cysteine, homocysteine, isocysteine, orpenicillamine; (amino acid)_(n) and (amino acid)_(m) are eachindependently any primary α- or β-amino acid that does not comprise athiol group; Z is --OH or --NH₂ ; and n and m are each independently aninteger between 2 and 5 and 0 and 5, respectively.

In the formulae describing the compositions of matter of the invention,the combination of the term "cyclo" and an underlined amino acidsequence will be understood to mean that the underlined amino acidsequence is cyclized through a peptide bond linking the amino terminusof the first amino acid of the sequence to the carboxyl terminus of thelast amino acid of the sequence. When followed by a parenthetical termsuch as "(CH₂ CO.X), said parenthetical term will be understood to meanthat the sequence in parentheses is covalently linked to the amino acidsidechain sulfur atom of the thiol-containing amino acid in theunderlined amino acid sequence, forming a sulfide bond therewith. Thus,in the above chemical structure, it will be understood that the --CH₂CO.X group is covalently linked to the sidechain sulfur atom ofhomocysteine. An example of such a chemical formula is shown in Example2.

A preferred embodiment of the compositions of matter of the invention isa compound having formula:

    cyclo(N-methyl)FYW.sub.D KV.Hcy.(CH.sub.2 CO.GGCK.amide).

The compositions of matter of the invention have utility for a number ofdiagnostic and therapeutic purposes.

Thus, one aspect of the invention provides reagents for preparingradiolabeled scintigraphic imaging agents for imaging sites within amammalian body having an overabundance of somatostatin receptors. Insuch embodiments, the metal ion-complexing moiety comprises a radiolabelbinding moiety, and the reagent forms a complex with the radiolabel viacomplex formation with said moiety. In one embodiment of this aspect ofthe invention, the scintigraphic imaging agent is formed by complexingthe reagent with Tc-99m under reducing conditions.

Thus, the invention also comprises scintigraphic imaging agents that arecomplexes of the reagents of the invention with Tc-99m and methods forradiolabeling the reagents. Tc-99m radiolabeled complexes provided bythe invention are formed by reacting the reagents of the invention withTc-99m in the presence of a reducing agent. Preferred reducing agentsinclude but are not limited to dithionite ion, stannous ion and ferrousion. Complexes of the invention are also formed by labeling the reagentsof the invention with Tc-99m by ligand exchange of a prereduced Tc-99mcomplex as provided herein.

A preferred embodiment of the reagents provided by the invention forpreparing scintigraphic imaging agents is a compound having formula:

    cyclo(N-methyl)FYW.sub.D KV.Hcy.(CH.sub.2 CO.GGCK.amide).

Preferred imaging agents comprise Tc-99m complexes of this reagent.

The invention also provides kits for preparing scintigraphic imagingagents that are the reagents of the invention radiolabeled with Tc-99m.Kits for labeling the reagents provided by the invention with Tc-99m arecomprised of a sealed vial containing a predetermined quantity of areagent of the invention and a sufficient amount of reducing agent tolabel the reagent with Tc-99m.

In a second aspect of the invention are provided imaging agents whereinthe compositions of matter of the invention are radiolabeled with aradioisotope of iodine, preferably I-123, I-125, or I-131. The inventionprovides radioiodinated imaging agents that are complexes of thereagents of the invention with radiolabeled iodine.

A preferred embodiment of the reagents provided by the invention forpreparing radioiodinated imaging agents is a compound having formula:

    cyclo(N-methyl)FYW.sub.D KV.Hcy.(CH.sub.2 CO.GGCK.amide).

Preferred imaging agents comprise radioiodinated embodiments of thisreagent.

In a third aspect are provided nonradioactively-labeled imaging agents,comprising reagents that are the compositions of matter of the inventioncomplexed with a paramagnetic metal ion or particle. In embodiments ofthis aspect of the invention, the paramagnetic metal ion is complexedwith the metal ion-complexing moiety. In embodiments where a particle,preferably a superparamagnetic metal particle, is used, the compositionsof matter of the invention are linked to the superparamagnetic particleeither covalently or associatively using methods described by Weisslederet al. (1992, Radiology 182: 381-385). The invention provides suchimaging agents for use in magnetic resonance imaging. Methods forpreparing these non-radiolabeled imaging agents are also provided.

A preferred embodiment of the reagents provided by the invention forpreparing such non-radioactively-labeled imaging agents is a compoundhaving formula:

    cyclo(N-methyl)FYW.sub.D KV.Hcy.(CH.sub.2 CO.GGCK.amide).

Preferred non-radioactively labeled imaging agents comprise ferric ioncomplexes of this reagent.

Therapeutic agents are also provided herein. In a fourth aspect, theinvention provides the compositions of matter of the inventionuncomplexed with a metal ion as a therapeutic agent per se. Preferredembodiments of such therapeutic agents provided by the invention arecompounds having formula:

    cyclo(N-methyl)FYW.sub.D KV.Hcy.(CH.sub.2 CO.GGCK.amide).

In a fifth aspect, the invention provides radiolabeled therapeuticagents comprising a reagent for preparing the therapeutic agent that isa composition of matter provided by the invention. In such embodiments,the compositions of matter of the invention are provided wherein themetal ion-complexing moiety is complexed with a β-particle emitting or aconversion electron emitting radioisotope. Preferred embodiments ofβ-particle emitting radioisotopes are rhenium-186 and rhenium-188. Apreferred conversion electron emitting radioisotope is tin-117m. Theinvention provides such radioisotope complexes of the compositions ofmatter of the invention for radiotherapeutic treatment ofsomatostatin-receptor expressing pathological cells and tissues,particularly neoplastic and metastatic cells. Methods for preparing suchtherapeutic radioisotope complexes of the compositions of matter of theinvention are also provided.

A preferred embodiment of the reagents of the invention provided forpreparing such radiotherapeutic agents is a compound having formula:

    cyclo(N-methyl)FYW.sub.D KV.Hcy.(CH.sub.2 CO.GGCK.amide).

Preferred radiotherapeutic agents comprise β-particle emitting orconversion electron-emitting metal ion complexes with this reagent.

In another aspect of the radiotherapeutic agents of the invention areprovided radiotherapeutic agents wherein the compositions of matter ofthe invention are radiolabeled with a radioisotope of iodine, preferablyI-125 or I-131, or with astatine-211. The invention also provides theradiolabeled therapeutic agents themselves, as well as methods forradiolabeling the reagents.

Preferred embodiments of the reagents of the invention provided forpreparing these therapeutic radioiodinated and radioastatinated agentsare compounds having formula:

    cyclo(N-methyl)FYW.sub.D KV.Hcy.(CH.sub.2 CO.GGCK.amide).

Preferred radiotherapeutic agents comprise radioiodinated andradioastatinated embodiments of this reagent.

Therapeutic agents are provided that are nonradioactively-labeled, metalatom-complexed agents, comprising reagents that are the compositions ofmatter of the invention complexed with a nonradioactive metal atom, suchas copper, zinc or rhenium. In embodiments of this aspect of theinvention, the nonradioactive metal ion is complexed with the metalion-complexing moiety. The invention provides such therapeutic agentsfor use in treating conditions in which somatostatin receptors areoverexpressed in the cells comprising certain tissues, and in treatingneoplastic cells which hyperexpress somatostatin receptors. Methods forpreparing these non-radiolabeled metal atom complexed therapeutic agentsare also provided.

A preferred embodiment of the reagents of the invention provided forpreparing non-radioactively-labeled therapeutic agents is a compoundhaving formula:

    cyclo(N-methyl)FYW.sub.D KV.Hcy.(CH.sub.2 CO.GGCK.amide).

Preferred non-radioactively labeled therapeutic agents comprise arhenium complex of this reagent.

This invention provides methods for preparing peptide embodiments of thereagents of the invention by chemical synthesis in vitro. In a preferredembodiment, peptides are synthesized by solid phase peptide synthesis.

This invention provides methods for using the diagnostic andradiodiagnostic and therapeutic and radiotherapeutic agents of theinvention. For radiolabeled embodiments of the agents of the invention,for example, Tc-99m labeled scintigraphic imaging agents, an effectivediagnostic or therapeutic amount of the diagnostic or radiodiagnostic ortherapeutic or radiotherapeutic agent of the invention are administered.In radiodiagnostic embodiments, localization of the radiolabel isdetected using conventional methodologies such as gamma scintigraphy. Innon-radioactive diagnostic embodiments, localization of sites ofaccumulation of the paramagnetic metal-labeled diagnostic agents of theinvention is achieved using magnetic resonance imaging methodologies.The imaging agents provided by the invention have utility for tumorimaging, particularly for imaging primary and metastatic neoplasticsites wherein said neoplastic cells express somatostatin receptors(SSTR), and in particular such primary and especially metastatic tumorcells that have been clinically recalcitrant to detection usingconventional methodologies. In addition, the imaging agents of theinvention are useful in detecting sites of T lymphocyte accumulationassociated with occult disease or pathology, e.g., as occurs in patientssuffering from tuberculosis.

The invention provides methods for using the somatostatin analogues ofthe invention to alleviate diseases or other ailments in animals,preferably humans. These diseases and ailments include but are notlimited to diabetes and diabetes-related retinopathy, cirrhosis of theliver and hepatitis infection, bleeding ulcers and othergastrointestinal bleeding, pancreatitis, central nervous systemdisorders, endocrine disorders, Alzheimer's disease, acromegaly andother diseases and disorders related to the production of inappropriatelevels of growth hormone in vivo, and cancer, particularly those cancerswhose growth is dependent or influenced by growth hormone production.Non-radiolabeled therapeutic embodiments also have utility for treatingSSTR hyperexpression-related ailments, such as diarrhea cause bySSTR-hyperexpressing gastrinoma. Dosages of the somatostatin analoguesprovided by the invention may be the same as those dosages of nativesomatostatin routinely used for treatment of the above or otherdiseases, or less of the compounds of the invention may be administereddue to their longer in vivo half-life.

Therapeutic uses also include radiotherapeutic ablation of neoplasticand metastatic malignant cells in patients bearing tumors the cells ofwhich express the somatostatin receptor. Use of the radiotherapeuticagents of the invention includes primary therapeutic use for tumorsrecalcitrant to more conventional therapies and for tumors that areinoperable, as well as adjunct therapies supplemental to surgery,radiation therapy or conventional chemotherapy.

The radiolabeled embodiments of the invention also have utility assurgical guides for identifying somatostatin receptor-expressing tumortissue during surgery. For such use in radioisotope guided surgery,malignant tissue otherwise invisible to the surgeon can be recognizedand excised during otherwise conventional surgery.

Specific preferred embodiments of the present invention will becomeevident from the following more detailed description of certainpreferred embodiments and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an image of ^(99m) Tc-labeled P587 in a tumor-bearing rat,indicated by an arrow, showing high uptake at the tumor site in thelower leg.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides cyclic peptides that are somatostatinanalogues and that do not comprise of a disulfide bond. Suchsomatostatin analogues thereby possess increased in vivo stabilitycompared with native somatostatin. These cyclic peptides are themselvestherapeutic agents for alleviating diseases and other ailments inanimals including humans.

Also provided by the invention are cyclic peptides that may beradioiodinated or radioastatinated and which are thereby useful inradiotherapeutic and radiodiagnostic applications.

Another embodiment of these cyclic peptides that is provided by thisinvention are cyclic peptide reagents wherein the cyclic peptides of theinvention are covalently linked to a metal ion-complexing moiety. Suchcyclic peptide reagents are capable of being radiolabeled to provideradiodiagnostic or radiotherapeutic agents. One example of aradiodiagnostic application using the radiolabeled agents of theinvention is scintigraphic imaging, wherein the location and extent ofsomatostatin receptor-bearing tumors may be determined. The cyclicpeptide reagents of the invention can also advantageously beradiolabeled with cytotoxic radioisotopes such as rhenium-186 orrhenium-188 for radiotherapeutic uses. The cyclic peptide reagents ofthe invention are also useful in preparing complexes withnon-radioactive metals, said complexes being useful therapeutically.

Labeling with Tc-99m is an advantage of the present invention becausethe nuclear and radioactive properties of this isotope make it an idealscintigraphic imaging agent. This isotope has a single photon energy of140 keV and a radioactive half-life of about 6 hours, and is readilyavailable from a ⁹⁹ Mo-^(99m) Tc generator. Other radionuclides may alsobe used in the practice of the invention as disclosed herein.

The term scintigraphic imaging agent as used herein is meant toencompass a radiolabeled agent capable of being detected with aradioactivity detecting means (including but not limited to agamma-camera, a Geiger-Muller counter and a scintillation detectorprobe).

Radiotherapeutic embodiments of the invention, on the other hand, areadvantageously labeled with a cytotoxic radioisotope, including but notlimited to copper-67, iodine-125, iodine-131, rhenium-186, rhenium-188,and astatine-211, most preferably ¹⁸⁶ Re or ¹⁸⁸ Re. Such embodiments areuseful in the treatment of somatostatin-related diseases or otherailments in animals, preferably humans, including but not limited tocancer and other diseases characterized by the growth of malignant orbenign tumors capable of binding somatostatin or somatostatin analoguesvia the expression of somatostatin receptors on the cell surface ofcells comprising such tumors.

The present invention provides reagents for preparing diagnostic andradiodiagnostic agents, and therapeutic and radiotherapeutic agents. Thereagents provided by the invention comprise a metal ion-complexingmoiety covalently linked to a specific binding peptide that binds tosomatostatin receptor sites within a mammalian body.

Small compounds, preferably having a molecular weight of less than10,000 daltons, are of distinct commercial advantage. Such smallcompounds can be readily manufactured. Moreover, they are likely not tobe immunogenic and to clear rapidly from the vasculature, thus allowingfor better and more rapid imaging. In contrast, larger molecules such asantibodies or fragments thereof, or other biologically-derived peptideslarger than 10,000 daltons, are costly to manufacture, and are likely tobe immunogenic and clear more slowly from the bloodstream, therebyinterfering with rapid diagnoses in vivo.

It is an advantage of the somatostatin analogues provided by thisinvention that the non-disulfide cyclic linkage contained therein isstable under the conditions of radiolabeling the covalently linkedradiolabel-binding moiety. In contrast, for example, Tc-99m conjugationto a Tc-99m binding moiety covalently linked to native somatostatin, orto a somatostatin analogue having a disulfide bond, can result inreduction of the disulfide accompanied by a loss of biological activity.Such loss of biological activity can also occur in vivo using nativesomatostatin, or to any somatostatin analogue having a disulfide bond.The present invention is not subject to similar losses in biologicalactivity in vivo because the non-disulfide cyclic linkages in each ofthe somatostatin analogues of the invention comprise stable covalentbonds.

For purposes of this invention, the term "specific binding peptide" isintended to mean any peptide compound that specifically binds to atarget site in a mammalian body defined by an overabundance ofsomatostatin receptor (SSTR) molecules. "Specific binding" will beunderstood by those with skill in this art as meaning that the peptidelocalizes to a greater extent at the target site that to surroundingtissues. Such specific binding is advantageous in diagnostic imagingagents comprising such specific binding peptides because they aredispersed throughout a mammalian body after administration and suchspecific localization of reagent-bound radioactivity provides visualdefinition of the target in vivo.

Each specific-binding peptide-containing embodiment of the invention iscomprised of a sequence of amino acids. The term amino acid as used inthis invention is intended to include all L- and D-, primary α- andβ-amino acids, naturally occurring, modified, substituted, altered andotherwise. Specific binding peptide embodiments of the reagents of theinvention comprise specific binding peptides having a molecular weightof less than about 5,000 daltons. Particularly preferred embodiments ofthe specific binding peptides of the invention include peptides thatbind specifically and with high affinity to the somatostatin receptor(SSTR) on SSTR-expressing cells, particularly tumor cells and activatedT-lymphocyte cells. Reagents comprising specific-binding peptidesprovided by the invention include but are not limited to reagentscomprising peptides having the following amino acid sequences (the aminoacids in the following peptides are L-amino acids except where otherwiseindicated):

cyclo(N-methyl)FYW_(D) KV.Hcy.(CH₂ CO.GGC.amide)

cyclo(N-methyl)FYW_(D) KV.Hcy.(CH₂ CO.GGCK.amide)

cyclo(N-methyl)FYW_(D) KV.Hcy.(CH₂ CO.GGCR.amide)

cyclo(N-methyl)FYW_(D) KV.Hcy.(CH₂ CO.GGCRD.amide)

cyclo(N-methyl)FYW_(D) KV.Hcy.(CH₂ CO.GGCRK.amide)

cyclo(N-methyl)FYW_(D) KV.Hcy.(CH₂ CO.GGCRR.amide)

cyclo(N-methyl)FYW_(D) KV.Hcy.(CH₂ CO.GGCKK.amide)

cyclo(N-methyl)FYW_(D) KV.Hcy.(CH₂ CO.GGCKKK.amide)

cyclo(N-methyl)FYW_(D) KV.Hcy.(CH₂ CO.GGC.Orn.amide)

cyclo(N-methyl)FYW_(D) KV.Hcy.(CH₂ CO.GGCKDK.amide)

cyclo(N-methyl)FYW_(D) KV.Hcy.(CH₂ CO.GGC.Orn.D.Orn.amide)

cyclo(N-methyl)FYW_(D) KV.Hcy.(CH₂ CO.GGC.Orn.D.amide)

cyclo(N-methyl)FYW_(D) KV.Hcy.(CH₂ CO.KKC.amide)

cyclo(N-methyl)FYW_(D) KV.Hcy.(CH₂ CO.KRC.amide)

cyclo(N-methyl)FYW_(D) KV.Hcy.(CH₂ CO.RRC.amide)

cyclo(N-methyl)FYW_(D) KV.Hcy.(CH₂ CO.KKCK.amide)

cyclo(N-methyl)FYW_(D) KV.Hcy.(CH₂ CO.GRCK.amide)

cyclo(N-methyl)FYW_(D) KV.Hcy.(CH₂ CO.GKCR.amide).

(Single-letter abbreviations for amino acids can be found in Zubay,ibid., p.33; other abbreviations are as in the Legend to Table I). Thislist of reagents provided by the invention is illustrative and notintended to be limiting or exclusive, and it will be understood by thosewith skill in the art that reagents comprising combinations of thepeptides disclosed herein or their equivalents may be covalently linkedto any of the chelating moieties of the invention and be within itsscope.

In a preferred embodiment, the reagent of the invention has formula:##STR2##

Specific-binding peptides comprising the reagents of the presentinvention can be chemically synthesized in vitro. Such peptides cangenerally advantageously be prepared on a peptide synthesizer. Thepeptides of this invention can be synthesized wherein the metalion-complexing moiety is covalently linked to the peptide duringchemical synthesis in vitro, using techniques taught herein (see, forexample, Example 2, subsection O).

In scintigraphic imaging embodiments of the invention, a complex oftechnetium-99m is formed with the reagents of this invention. Toaccomplish this, Tc-99m, preferably as a salt of Tc-99m pertechnetate,is reacted with the reagents of this invention in the presence of areducing agent. Preferred reducing agents are dithionite, stannous andferrous ions; the most preferred reducing agent is a stannous salt suchas stannous chloride. In an additional preferred embodiment, thereducing agent is a solid-phase reducing agent. Complexes and means forpreparing such complexes are conveniently provided in a kit formcomprising a sealed vial containing a predetermined quantity of areagent of the invention to be labeled and a sufficient amount ofreducing agent to label the reagent with Tc-99m. Alternatively, thecomplex may be formed by reacting a reagent of this invention with apre-formed labile complex of technetium and another compound known as atransfer ligand (such as tartrate, citrate, gluconate or mannitol, forexample). This process is known as ligand exchange and is well known tothose skilled in the art. Among the Tc-99m pertechnetate salts usefulwith the present invention are included the alkali metal salts such asthe sodium salt, or ammonium salts or lower alkyl ammonium salts.

Technetium-99m labeled scintigraphic imaging agents according to thepresent invention can be prepared by the addition of an appropriateamount of Tc-99m or Tc-99m complex into the vials and reaction underconditions described in Example 3 herein below. The kit may also containconventional pharmaceutical adjunct materials such as, for example,pharmaceutically acceptable salts to adjust the osmotic pressure,buffers, preservatives and the like. The components of the kit may be inliquid, frozen or dry form. In a preferred embodiment, kit componentsare provided in lyophilized form. Radiolabeled scintigraphic imagingreagents according to the present invention may be prepared by reactionunder conditions described in Example 3 herein below.

Radioactively labeled reagents provided by the present invention areprovided having a suitable amount of radioactivity. In forming, forexample, Tc-99m radioactive complexes, it is generally preferred to formradioactive complexes in solutions containing radioactivity atconcentrations of from about 0.01 millicurie (mCi) to 100 mCi per mL.

The imaging reagents provided by the present invention can be used forvisualizing organs such as the kidney for diagnosing disorders in theseorgans, and tumors, in particular gastrointestinal tumors, myelomas,small cell lung carcinoma and other APUDomas, endocrine tumors such asmedullary thyroid carcinomas and pituitary tumors, brain tumors such asmeningiomas and astrocytomas, and tumors of the prostate, breast, colon,and ovaries can also be imaged. In accordance with this invention, theTc-99m labeled peptide reagents are administered in a single unitinjectable dose. The Tc-99m labeled peptide reagents provided by theinvention may be administered intravenously in any conventional mediumfor intravenous injection such as an aqueous saline medium, or in bloodplasma medium. Generally, the unit dose to be administered has aradioactivity of about 0.01 mCi to about 100 mCi, preferably 1 mCi to 20mCi. The solution to be injected at unit dosage is from about 0.01 mL toabout 10 mL. After intravenous administration, imaging in vivo can takeplace in a matter of a few minutes. However, imaging can take place, ifdesired, in hours or even longer, after the radiolabeled peptide isinjected into a patient. In most instances, a sufficient amount of theadministered dose will accumulate in the area to be imaged within about0.1 of an hour to permit the taking of scintiphotos. Any conventionalmethod of scintigraphic imaging for diagnostic purposes can be utilizedin accordance with this invention.

The somatostatin receptor-binding cyclic peptides and non-radioactivemetal complexes of the cyclic peptide reagents of the invention may beused clinically as therapeutic agents to promote regression of certaintypes of tumors, particularly those that express somatostatin receptors.The somatostatin analogue cyclic peptides of the invention can also beused to reduce the hormonal hypersecretion that often accompaniescertain cancers, such as the APUDomas. Peptides of the invention used astherapeutic agents may be administered by any appropriate route,including intravenous, intramuscular or by mouth, and in any acceptablepharmaceutical carrier, in doses ranging from about 0.1 to about 49mg/kg body weight/day.

This invention also provides peptides radiolabled with cytotoxicradioisotopes such as rhenium-186 or rhenium-188 that may be used forradiotherapy of certain tumors as described above. For this purpose, anamount of radioactive isotope from about 10 mCi to about 200 mCi may beadministered via any suitable clinical route, preferably by intravenousinjection.

The methods for making and labeling these compounds are more fullyillustrated in the following Examples. These Examples illustrate certainaspects of the above-described method and advantageous results, and areshown by way of illustration and not limitation.

EXAMPLE 1

Solid Phase Peptide Synthesis

Solid phase peptide synthesis (SPPS) was carried out on a 0.25 millimole(mmole) scale using an Applied Biosystems Model 431A Peptide Synthesizerand using 9-fluorenylmethyloxycarbonyl (Fmoc) amino-terminus protection,coupling with dicyclohexylcarbodiimide/hydroxybenzotriazole or2-(1H-benzo-triazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate/hydroxybenzotriazole (HBTU/HOBT), and usingp-hydroxymethyl-phenoxymethylpolystyrene (HMP) or Sasrin™ resin forcarboxyl-terminus acids or Rink amide resin for carboxyl-terminusamides.

Fmoc.Hcy(S-trityl) and Fmoc.Pen(S-trityl) were prepared from theappropriate amino acid by tritylation with triphenylmethanol intrifluoroacetic acid, followed by Fmoc derivitization as described byAtherton et al. (1989, Solid Phase Peptide Synthesis, IRL Press: Oxford)and further in Example 2, subsection J below.Fmoc-S-(3-Boc-aminopropyl)cysteine was prepared from L-cysteine andBoc-aminopropyl bromide in methanolic sodium methoxide followed bytreatment with O-9-fluorenylmethyl-O'-N-succcinimidyl carbonate(FmocOSu) at pH 10.

2-haloacetyl groups were introduced either by using the appropriate2-haloacetic acid as the last residue to be coupled during SPPS or bytreating the N-ternminus free amino peptide bound to the resin witheither 2-haloacetic acid/diisopropylcarbodiimide/N-hydroxysuccinimide inNMP of 2-haloacetic anhydride/diisopropylethylamine in NMP.

Thiol-containing peptides were reacted with chloroacetyl-containing,thiol-protected Tc-99m complexing moieties at pH 10 for 0.5-24 hours atroom temperature, optionally followed by acetic acid acidification andevaporation of the solution to give the corresponding peptide-sulfideadduct, as described in more detail in Example 2, subsection O below.Deprotection and purification were routinely performed as described toyield the chelator-peptide conjugate.

Sasrin™ resin-bound peptides were cleaved using a solution of 1% TFA indichloromethane to yield the protected peptide.

Where appropriate, protected peptide precursors were cyclized betweenthe amino- and carboxyl-termini by reaction of sidechain-protected,amino-terminal free amine and carboxyl-terminal free acid usingdiphenylphosphorylazide, as described in more detail in Example 2,subsection M below.

HMP or Rink amide resin-bound products were routinely cleaved andprotected cyclized peptides deprotected using a solution comprised oftrifluoroacetic acid (TFA), or TFA and methylene chloride, optionallycomprising water, thioanisole, ethanedithiol, and triisopropylsilane inratios of 100:5:5:2.5:2, for 0.5-3 hours at room temperature. Whereappropriate, products were re-S-tritylated in triphenolmethanol/TFA, andN-Boc groups re-introduced into the peptide using (Boc)₂ O.

Crude peptides were purified by preparative high pressure liquidchromatography (HPLC) using a Waters Delta-Pak C18 column and gradientelution with 0.1% TFA in water modified with acetonitrile. After columnelution, acetonitrile was evaporated from the eluted fractions, whichwere then lyophilized. The identity of each product so produced andpurified was confirmed by fast atom bombardment mass spectroscopy(FABMS) or electrospray mass spectroscopy (ESMS).

EXAMPLE 2

Synthesis ofcyclo(N-CH₃)phenylalanyl-tyrosyl-D-tryptophyl-lysyl-valyl-homocysteine,S-2-acetyl-glycyl-glycyl-cysteinyl-lysinamide (P587)

A. Synthesis of N-α-carbobenzoxy-N-ε-tert-butoxycarbonyl-lysine,N-hydroxy-succinimide ester

To a mixture of N-α-carbobenzoxy-N-ε-tert-butoxycarbonyl lysine (23 g,60.5 mmol) and N-hydroxysuccinimide (7.1 g, 61.7 mmol) in 180 mL drytetrahydrofuran (THF) cooled in a cold water bath was addeddiisopropylcarbodiimide (9.66 mL, 61.7 mmol). This reaction mixture wasstirred overnight and then filtered and the filtrate evaporated. Theresidue of the filtrate was then redissolved in minimal ethyl acetate,200 mL ether and 200 mL hexanes. The title compound precipitated fromthis mixture and was recovered by filtration, washed with cold hexanesand dried to give 28.1 g (58.9 mmol, 97% yield).

B. Synthesis of N-α-carbobenzoxy-N-ε-tert-butoxycarbonyl-lysyl-valine,methyl ester

To a solution of valine methyl ester (8.38 g, 50 mmol) anddiisopropylethylamine (12.7 mL, 50 mmol) in 150 mL THF was addedN-α-carbobenzoxy-N-ε-tert-butoxycarbonyl lysine, N-hydroxysuccinimideester (23.9 g, 50 mmol), followed by an additional 50 mL THF. After 2 hthe solvent was removed by evaporation and 50 mL ethyl acetate added tothe residue. This solution was washed sequentially with 200 mL 5% citricacid, 200 mL saturated sodium bicarbonate and 200 mL saturated brine,dried over anhydrous magnesium sulfate, filtered and evaporated. Theresulting oil was dissolved in minimal ethyl acetate, 200 mL ether and200 mL hexanes. The title compound was isolated by filtration and driedto yield 20 g (40.5 mmol, 81% yield).

C. Synthesis of N-ε-tert-butoxycarbonyl-lysyl-valine, methyl ester

To a solution of N-α-carbobenzoxy-N-ε-tert-butoxycarbonyl-lysyl-valine,methyl ester (19 g, 38.5 mmol) and acetic acid (1 mL) in 100 mL ethylacetate was added 10% palladium on carbon catalyst (0.19 g) and stirredunder a hydrogen atmosphere overnight. The reaction mixture was thenfiltered over Celite, the filtrate evaporated and the residueredissolved in 100 mL methanol containing 1 mL acetic acid. To thissolution was added 10% palladium on carbon (0.19 g) and the mixture washydrogenated under a pressure of 45 pounds per square inch for 2 h usinga Parr hydrogenator. The reaction mixture was again filtered over Celiteand the filtrate evaporated to give 13.56 g of the title compound (37.7mmol, 98% yield).

D. Synthesis of fluorenylmethoxycarbonyl-D-tryptophan,N-hydroxysuccinimide ester

To a solution of N-α-fluorenylmethoxycarbonyl-tryptophan hemihydrate (25g, 82.1 mmol) and N-hydroxysuccinimide (9.78 g, 85 mmol) in 250 mL dryTHF cooled in a cold water bath was added diisopropylcarbodiimide (13.3mL, 85 mmol). The reaction mixture was stirred overnight, filtered andthe filtrate evaporated. The residue was then redissolved in toluene andhexanes. The title compound was isolated by filtration and dried toyield 34 g (66.5 mmol, 81% yield).

E. Synthesis offluorenylmethoxycarbonyl-D-tryptophyl-N-ε-tert-butoxy-carbonyl-lysyl-valine,methyl ester

To a mixture of N-ε-tert-butoxycarbonyl-lysyl-valine, methyl ester (13g, 36.1 mmol) in 200 mL THF was addedfluorenylmethoxycarbonyl-D-tryptophan, N-hydroxysuccinimide ester (18.9g, 36.1 mmol). Diisopropylethylamine was added to adjust the pH of thereaction mixture to pH 8, and the reaction stirred for 2 days. Thesolvent was then removed by evaporation and the residue redissolved inethyl acetate, washed with 5% citric acid, saturated bicarbonate, andsaturated brine, and then dried over sodium sulfate. The solution wasthen filtered and evaporated and the residue redissolved in ethylacetate. The title compound was recovered from several crystallizationsof this solution to give 17.3 g of product (22.5 mmol, 62% yield).

F. Synthesis of N-fluorenylmethoxycarbonyl-O-tert-butyltyrosine,N-hydroxy-succinimide ester

To a solution of N-α-fluorenylmethoxycarbonyl-O-tert-butyltyrosine (25g, 54.3 mmol) and N-hydroxysuccinimide (6.62 g, 57.5 mmol) in 250 mL dryTHF cooled in a cold water bath was added diisopropylcarbodiimide (9.0mL, 57.5 mmol). The reaction mixture was stirred overnight, filtered andthe filtrate evaporated. The residue was then redissolved in toluene andhexanes. The title compound was isolated by filtration and dried toyield 27.7 g (49.7 mmol, 92% yield).

G. Synthesis ofN-fluorenylmethoxycarbonyl-O-tert-butyltyrosyl-D-tryptophyl-N-ε-tert-butoxycarbonyl-lysyl-valine,methyl ester

Fluorenylmethoxycarbonyl-D-tryptophyl-N-ε-tert-butoxycarbonyl-lysyl-valine,methyl ester (17 g, 22.1 mmol) was treated with 40 mL diethylamine and40 mL THF at room temperature for 1.5 h. The reaction mixture was thenevaporated, resuspended in 100 mL THF and re-evaporated three times. Theresidue was taken up in 120 mL dry THF andN-fluorenylmethoxycarbonyl-O-tert-butyltyrosine, N-hydroxysuccinimideester (12.3 g, 22.1 mmol) was added, followed by 20 mL dry THF and 4 mLdiisopropylethylamine resulting in a solution having a pH of 9. Afterstirring overnight, the reaction was evaporated and the residue taken upin ethyl acetate. The ethyl acetate solution was washed with 5% citricacid, saturated sodium bicarbonate and saturated brine, then dried overmagnesium sulfate and evaporated. The residue was taken up in ethylacetate, ether and hexanes and the title compound precipitated. Theprecipitated compound was isolated by filtration and dried to yield 14.6g of the title compound (14.8 mmol, 67% yield).

H. Synthesis of N-fluorenylmethoxycarbonyl-N-methylphenylalanine,N-hydroxysuccinimide ester

To a solution of N-α-fluorenylmethoxycarbonyl-N-methylphenylalanine (25g, 62.3 mmol) and N-hydroxysuccinimide (7.5 g, 65 mmol) in 180 mL dryTHF cooled in a cold water bath was added diisopropylcarbodiimide (10mL, 64.2 mmol). The reaction mixture was stirred overnight, filtered andthe filtrate evaporated. The residue was then redissolved in toluene andhexanes. The title compound was isolated by filtration and dried toyield 28.3 g (57 mmol, 91% yield).

I. Synthesis ofN-fluorenylmethoxycarbonyl-N-methylphenylalanine-O-tert-butyl-tyrosyl-D-tryptophyl-N-ε-tert-butoxycarbonyl-lysyl-valine,methyl ester

Fluorenylmethoxycarbonyl-O-tert-butyl-tyrosyl-D-tryptophyl-N-ε-tert-butoxycarbonyl-lysyl-valine,methyl ester (14 g, 14.2 mmol) was treated with 35 mL diethylamine and35 mL THF at room temperature for 1 h. The reaction mixture was thenevaporated, resuspended in 100 mL THF and re-evaporated three times. Theresidue was taken up in ethyl acetate and hexanes. The product,O-tert-butyltyrosyl-D-tryptophyl-N-ε-tert-butoxycarbonyl-lysyl-valine,methyl ester was precipitated, isolated by filtration and dried to yield9.7 g (12.7 mmol, 90% yield). TheO-tert-butyltyrosyl-D-tryptophyl-N-ε-tert-butoxycarbonyl-lysyl-valine,methyl ester was dissolved in 35 mL dry THF andN-fluorenylmethoxycarbonyl-N-methylphenylalanine, N-hydroxysuccinimideester (6.35 g, 14.2 mmol) was added, followed by 3 mLdiisopropylethylamine resulting in a solution having a pH of 9. Afterstirring overnight, the reaction was evaporated and the residue taken upin ethyl acetate. The ethyl acetate solution was washed with 5% citricacid, saturated sodium bicarbonate and saturated brine, then dried overmagnesium sulfate and evaporated. The residue was taken up in ethylacetate, ether and hexanes and the title compound precipitated. Theprecipitated compound was isolated by filtration and dried to yield 12.4g of the title compound (11.5 mmol, 81% yield).

J. Synthesis of N-fluorenylmethoxycarbonyl-S-tritylhomocysteinie

a. To about 400 mL liquid ammonia cooled to -78° C. in a dry ice/ethanolbath was added small amounts of elemental sodium followed by asufficient amount of homocysteine to quench the resulting blue color ofthe sodium/ammonia solution until 5.7 g (248 mmol) sodium and 9.75 g(36.3 mmol) homocysteine had been consumed and the blue color of thesodium/ammonia solution persisted for about 15 min. Ammonium chloride(0.5 g) was added to quench the final blue color, and then the reactionwas removed from the cooling bath and the ammonia allowed to evaporateunder an argon stream. The flask was warmed slightly to drive offessentially all residual ammonia.

To the residue was added triphenylmethanol (23.6 g, 91 mmol) and thereaction flask cooled in an ice/water bath. 250 mL trifluoroacetic acid(TFA) was added, and after 30 min the mixture was evaporated and theresidue redissolved and re-evaporated three times with chloroform. Theresidue was then redissolved in 300 mL water and the pH adjusted to pH 4with 5% citric acid and 1M KOH. The product precipitated as a gum andwas collected by filtration. The residue was then triturated with etherto yield S-trityl-homocysteine (7 g, 18.6 mmol, 26% yield). A secondcrop was isolated from the filtrate by crystallization fromdimethylformamide (DMF)/water to give a combined yield of 24.2 g (64mmol, 89%).

b. To a solution of S-trityl-homocysteine (20 g, 53 mmol) in 150 mLacetone/100 mL water was added sodium carbonate (11.5 g, 109 mmol) andthen O-fluorenylmethyl-O'-(N-succinimidyl)carbonate (17.5 g, 52 mmol)dissolved in 200 mL acetone, these additions being made over the courseof about 1 h. The reaction mixture was then stirred for about 2 days andthen the organic solvents were evaporated. To the aqueous residue wasadded 300 mL ethyl acetate and the mixture was acidified with 1M HCl.The organic phase was separated and washed sequentially with 1M HCl,0.5M HCl, and 0.25M HCl, then dried over magnesium sulfate, filtered andevaporated. The crude product was chromatographed on silica gel (100%chloroform→3% methanol in chloroform) to yield the title compound (19.4g, 32 mmol, 62% yield).

K. Synthesis ofN-fluorenylmethoxycarbonyl-S-trityl-homocysteine-N-methyl-phenylalanine-O-tert-butyltyrosyl-D-tryptophyl-N-ε-tert-butoxycarbonyl-lysyl-valine,methyl ester

Fluorenylmethoxycarbonyl-N-methylphenylalanine-O-tert-butyl-tyrosyl-D-tryptophyl-N-ε-tert-butoxycarbonyl-lysyl-valine,methyl ester (9.8 g, 8.5mol) was treated with 28 mL diethylamine and 30mL THF at room temperature for 1 h. The reaction mixture was thenevaporated, resuspended in 100 mL THF and re-evaporated three times. Theresidue was taken up in ethyl acetate and hexanes. The product,N-methylphenylalanine-O-tert-butyltyrosyl-D-tryptophyl-N-ε-tert-butoxycarbonyl-lysyl-valine,methyl ester was precipitated, isolated by filtration and dried to yield4.9 g (8.1 mmol, 95% yield).

To a solution of N-fluorenylmethoxycarbonyl-S-trityl-homocysteine in 50mL dry THF cooled in a -15° C. cooling bath was addedN,N'-bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-Cl; 2.47 g, 9.7mmol) and 1.7 mL diisopropylethylamine (9.7 mmol) and the reactionmixture was stirred for 30 min.N-methylphenylalanine-O-tert-butyltyrosyl-D-tryptophyl-N-ε-tert-butoxycarbonyl-lysyl-valine,methyl ester (7.48 g) in 50 mL dry THF was added, followed by anadditional 1.7 mL diisopropylethylamine. The reaction volume was reduced50% by evaporation and then stirred for 2 days at room temperature. Thesolvent was removed by evaporation and the residue redissolved in ethylacetate, washed with 5% citric acid, saturated sodium bicarbonate andsaturated brine, and dried over magnesium sulfate. The residue was takenup in ethyl acetate, ether and hexanes and the title compoundprecipitated. The precipitated compound was isolated by filtration togive 10.2 g (6.77 mmol, 84% yield).

L. Synthesis ofS-trityl-homocysteine-N-methylphenylalanine-O-tert-butyl-tyrosyl-D-tryptophyl-N-ε-tert-butoxycarbonyl-lysyl-valine

A solution ofN-fluorenylmethoxycarbonyl-S-trityl-homocysteine-N-methylphenylalanine-O-tert-butyl-tyrosyl-D-tryptophyl-N-ε-tert-butoxycarbonyl-lysyl-valine,methyl ester (10 g, 6.6 mmol) and LiOH.2H₂ O in 50 mL THF and 4 mL waterwas stirred for 3 days. An additional 25 mol % LiOH.2H₂ O was added, andtwo hours later the solvent was evaporated and the residue redissolvedin ethyl acetate. This solution was then washed with 5% citric acid,saturated sodium bicarbonate, and saturated brine, dried over magnesiumsulfate, filtered and evaporated. The residue was redissolved in ethylacetate, ether and hexanes and the title compound precipitated and wasisolated by filtration and dried. The resulting impure product was flashchromatographed in silica gel using chloroform/10% methanol inchloroform to yield 3.46 g of the pure title compound (47 mmol, 41%yield).

M. Synthesis ofcyclo-N-methylphenylalanine-O-tert-butyltyrosyl-D-tryptophyl-N-ε-tert-butoxcarbonyl-lysyl-valyl-S-trityl-homocysteine

A solution ofS-trityl-homocysteine-N-methylphenylalanine-O-tert-butyl-tyrosyl-D-tryptophyl-N-ε-tert-butoxycarbonyl-lysyl-valine(3.42 g, 2.69 mmol) dissolved in 1740 mL DMF was cooled in an ice waterbath, and 1.16 mL diisopropylethylamine and diphenyl phosphorylazide(DPPA; 5.4 g, 1.16 mmol) were added. The reaction was incubated at -20°C. for 5 days and an additional 25 mol % DPPA was added, followed by anadditional 100 mol % diisopropylethylamine. The DMF solvent was thenremoved by evaporation and the crude title compound crystallized fromethyl acetate, ether and hexanes to yield 2.43 g (1.9 mmol, 69%).

N. Synthesis ofcyclo-N-methylphenylalanyl-tyrosyl-D-tryptophyl-lysyl-valyl-S-trityl-homocysteine

cyclo-S-trityl-homocysteine-N-methylphenylalanine-O-tert-butyl-tyrosyl-D-tryptophyl-N-ε-tert-butoxycarbonyl-lysyl-valine(2.34 g, 1.9 mmol) was treated with 18.7 mL TFA, 2 mL dichloromethane,0.94 mL water, 0.47 mL ethanedithiol and 0.37 mL triisopropylsilane for1 h at room temperature. TFA was removed by evaporation and the residueredissolved and reevaporated from chloroform three times. The residuewas then redissolved in 10 mL chloroform and poured into 400 mL coldether. The crude product precipitate was collected by filtration andpurified by C18 preparative reverse phase HLPC (using a gradient of 30%acetonitrile→60% acetonitrile in water, all solvents containing 0.1%TFA) to give the title compound (1 g, 1.2 mmol, 62% yield). Fast atombombardment mass spectrometry (FABMS) analysis gave a MH⁺ of 855,compared with the theoretical (average) predicted value of 855.09.

O. Synthesis ofcyclo-N-methylphenylalanyl-tyrosyl-D-tryptophyl-lysyl-valyl-S-trityl-homocysteine,S-2-acetyl-glycyl-glycyl-cysteinyl-lysinamide (P587)

cyclo-N-methylphenylalanyl-tyrosyl-D-tryptophyl-lysyl-valyl-homocysteine(250 mg, 0.29 mmol) and2-chloroacetyl-glycyl-glycyl-S-trityl-cysteinyl-lysylamide (prepared bySPPS as described in Example 1; 238 mg, 0.35 mmol) were dissolved in 7mL, acetonitrile and 7 mL of a solution of 100 mM sodium carbonate/0.5mM EDTA, pH10 and stirred overnight. The reaction mixture was thenevaporated to dryness and deprotected in TFA containingtriisopropylsilane as described above in Example 1. The title compoundwas then purified by reverse phase HLPC to yield 92.4% of the expectedtheoretical yield. Fast atom bombardment mass spectrometry analysis gavea MH⁺ of 1258, compared with the theoretical (average) predicted valueof 1257.70. The structure of this product, termed P587, is representedby the formula: ##STR3##

EXAMPLE 3

General Methods for Radiolabeling

0.1 mg of a peptide reagent prepared as in Example 1 was dissolved in0.1 mL of water, or 0.9% sodium chloride, or 10%hydroxypropylcyclodextrin (HPCD), or 50:50 ethanol:water, orphosphate-buffered saline (PBS), or 50 mM potassium phosphate buffer(pH=5, 6 or 7.4). Tc-99m gluceptate was prepared by reconstituting aGlucoscan vial (E.I. DuPont de Nemours, Inc., Wilmington, Del.) with 1.0mL of Tc-99m sodium pertechnetate containing up to 200 mCi and allowedto stand for 15 minutes at room temperature. 25 μL of Tc-99m gluceptatewas then added to the reagent and the reaction allowed to proceed atroom temperature or at 100° C. for 5-30 min and then filtered through a0.2 μm filter.

The Tc-99m labeled peptide reagent purity was determined by HPLC usingthe following conditions: a Waters Delta-Pak RP-18 analytical column,having dimensions of 5 μm×4.6 mm×220 mm, was loaded with eachradiolabeled peptide, which were then eluted at a solvent flow rate of 1mL/min. Gradient elution was performed over 10-20 min using a lineargradient beginning with 100% Solvent A (0.1% TFA/water) and ending with100% Solution B (0.1% TFA/90% acetonitrile/water). Radioactivecomponents were detected by an in-line radiometric detector linked to anintegrating recorder. Tc-99m gluceptate and Tc-99m sodium pertechnetateelute between 1 and 4 minutes under these conditions, whereas the Tc-99mlabeled peptide eluted after a much greater amount of time.

The following Table illustrates successful Tc-99m labeling of peptidesprepared according to Example 1 using the method described herein.

                                      TABLE I                                     __________________________________________________________________________                             FABMS                                                                              Radiochemical                                                                        HPLC                                       Peptides MH.sup.+ Yield(%)* R.sub.T (min)                                   __________________________________________________________________________    cyclo(N-methyl)FYW.sub.D KV.Hcy.(CH.sub.2 CO.GGC.amide)                                                1129 98.sup.2                                                                             15.1, 17.2                                 cyclo(N-methyl)FYW.sub.D KV.Hcy.(CH.sub.2 CO.GGCK.amide) 1258 99.sup.2                                           15.0                                       cyclo(N-methyl)FYW.sub.D KV.Hcy.(CH.sub.2 CO.GGCR.amide) 1285 99.sup.1                                           15.1                                       cyclo(N-methyl)FYW.sub.D KV.Hcy.(CH.sub.2 CO.GGCKK.amide) 1386 N.D.                                              N.D.                                       cyclo(N-methyl)FYW.sub.D KV.Hcy.(CH.sub.2 CO.GGC.Orn.amide) 1244                                                 98.sup.3 7.0                             __________________________________________________________________________     *Superscripts refer to the following labeling conditions:                     .sup.1 = in 10% HPCD at room temperature                                      .sup.2 = in 50/50 ethanol/water at room temperature                           .sup.3 = in 0.9% NaCl at 100° C.                                  

HPLC methods (indicated by superscript after R_(T)):

Waters-1 column, 100% Solution A→100% Solution B in 10 min.

Single-letter abbreviations for amino acids can be found in Zubay, ibid.p.33. Underlining indicates the formation of an amide or a thiol linkagebetween the linked amino acids of derivative groups. Acm isacetamidomethyl; Orn is ornithine; F_(D) is D-phenylalanine; Y_(D) isD-tyrosine; W_(D) is D-trytophan; Apc=L-(S-(3-aminopropyl)cysteine; andHcy is homocysteine.

Radioiodination and radioastatination are performed as described bySeevers et al. (1982, Chem. Rev. 82: 575-590).

Non-radioactive rhenium complexes were prepared by co-dissolving each ofthe peptide reagents of the invention with about one molar equivalent oftetrabutylammonium oxotetra-bromorhenate (+5), prepared as described byCotton et al. (1966, Inorg. Chem. 5: 9-16) in dimethylformamide oracetonitrile/water and stirred for 0.5-5 days. The rhenium complexeswere isolated by reverse phase HPLC as described above for Tc-99mlabeled peptides and were characterized by FABMS or ESMS.

Radioactive rhenium complexes, using for example Re-186 or Re-188, areprepared from the appropriate perrhenate salts using the same protocolas for Tc-99m labeling, or by adding a reducing agent to a solution ofthe peptide and perrhenate, or optionally using a ligand transfer agentsuch as citrate and incubating the reaction at a temperature betweenroom temperature and 100° C. for between 5 and 60 min.

EXAMPLE 4

Inhibition of (¹²⁵ I-Tyr¹¹)Somatostatin-14 Binding to AR42J RatPancreatic Tumor Cell Membranes

The ability of various somatostatin receptor-binding reagents of theinvention to bind to somatostatin receptors in vitro was demonstrated inan assay of peptide reagent-mediated inhibition of binding of aradiolabeled somatostatin analogue to somatostatin receptor-containingcell membranes.

The rat pancreatic tumor cell line AR42J expressing the sonmatostatinreceptor was cultured in Dulbecco's modified essential media (DMEM)supplemented with 10% fetal calf serum (FCS) and 8 mM glutamine in ahumidified 5% CO₂ atmosphere at 37° C. Harvested cells were homogenizedin cold buffer (50 mM Tris-HCl, pH 7.4), and the homogenate was thencentrifuged at 39,000 g for 10 min at 4° C. Pellets were washed oncewith buffer and then resuspended in ice-cold 10 mM Tris-HCl buffer (pH7.4). Equal aliquots of this cell membrane preparation were thenincubated with (¹²⁵ I-Tyr¹¹)somatostatin-14 (Amersham, ArlingtonHeights, Ill.) at a final concentration of 0.5 nM at 750,000cpm/mL,specific activity 2000 Ci/mmol and either a peptide or peptide-rheniumcomplex of the invention (at a final concentration ranging from 10⁻¹¹ Mto 10⁻⁶ M in 50 mM HEPES buffer, pH 7.4, containing 1% bovine serumalbumin, 5 mM MgCl₂, 0.02 mg/mL bacitracin, 0.02 mg/mLphenylmethyl-sulfonylfluoride and 200,000 IU Trasylol for 25 min at 30°C.

After incubation, this membrane mixture was filtered through apolyethyleneimine-washed GC/F filter (Whatman Ltd., Maidstone, England)using a filtration manifold, and the residue remaining on the filter waswashed three times with 5 mL cold HEPES buffer. The filter and a sampleof the filter washings were then counted on a gamma counter. To assessnon-specific binding, the assay was also performed essentially asdescribed in the presence of 200 mn unlabeled somatostatin-14. Dataanalysis included Hill plots of the data to yield inhibition constantsas described by Bylund and Yamamura (1990, Methods in Neuro-transmitterReceptor Analysis, Yamamura et al., eds., Raven Press: New York). Theresults obtained using this assay with the reagents of the invention areas follows:

                  TABLE II                                                        ______________________________________                                        Peptide                    K.sub.i (nM)                                       ______________________________________                                        cyclo(N-methyl)FYW.sub.D KV.Hcy.(CH.sub.2 CO.GGCKK.amide)                                                0.26                                                 cyclo(N-methyl)FYW.sub.D KV.Hcy.(CH.sub.2 CO.GGCK.amide) 2.5                  cyclo(N-methyl)FYW.sub.D KV.Hcy.(CH.sub.2 CO.GGC.amide) 2.6                 ______________________________________                                    

These results demonstrate that peptide reagents of the invention bindwith high affinity to somatostatin receptors in vitro.

EXAMPLE 5

Localization and In Vivo Imaging of Somatostatin Receptor(SSTR)-Expressing Tumors in Rats

In vivo imaging of somatostatin receptors expressed by rat tumor cellswas performed essentially as described by Bakker et al. (1991, LifeSciences 49: 1593-1601).

CA20948 rat pancreatic tumor cells, thawed from frozen harvested tumorbrei, were implanted intramuscularly in a suspension of 0.05 to 0.1mL/animal, into the right hind thigh of 6 week old Lewis rats. Thetumors were allowed to grow to approximately 0.5 to 2 g, harvested, andtumor brei was used to implant a second, naive set of Lewis rats.Passaging in this fashion was repeated to generate successivegenerations of tumor-bearing animals. The tumor-bearing animals used forthe in vivo studies were usually from the third to fifth passage andcarried 0.2 to 2 g tumors.

For studies of the specificity of radiotracer localization in thetumors, selected animals were given an subcutaneous SSTR-blocking dose(4 mg/kg) of octreotide 30 minutes prior to injection of theradiotracer. (This protocol has been shown by Bakker et al. to result ina lowering of ¹¹¹ In-(DTPA)octreotide tumor uptake by 40%.)

Third- to fifth-passage CA20948 tumor-bearing Lewis rats were restrainedand injected intravenously via the dorsal tail vein with a dose of0.15-0.20 mCi ^(99m) Tc-labeled peptide corresponding to 3 to 8 μgpeptide in 0.2 to 0.4 mL.

At selected times, the animals were sacrificed by cervical dislocationand selected necropsy was performed. Harvested tissue samples wereweighed and counted along with an aliquot of the injected dose in agamma well-counter.

The 90-minute biodistribution results of selected radiolabeled peptidesare presented in Table I. Notably, ^(99m) Tc-P587, ^(99m) Tc-P617,^(99m) Tc-P726, and ^(99m) Tc-P736 showed very high tumor uptake andtumor/blood ratios demonstrating their high specific uptake in target(tumor) tissue.

FIG. 1 shows an image of ^(99m) Tc-P587 in a tumor-bearing rat. The highuptake in the tumor in the lower leg (arrow) is clearly visible.

^(99m) Tc-P587 uptake in tumors in rats was compared with and withoutpre-injection treatment with octreotide, a somatostatin analogue knownto bind to the somatostatin receptor in vivo. In these experiments,receptor-blocking by administration of octreotide prior toadministration of ^(99m) Tc-P587 reduced specific tumor uptake of theradiolabeled peptide by 76%. These results confirmed that binding of^(99m) Tc-P587 in vivo was SSTR-specific.

                                      TABLE III                                   __________________________________________________________________________                                % ID/g                                            No.                                                                              Peptides                 Tumor                                                                             Blood                                                                             Tumor/Blood                               __________________________________________________________________________    P736                                                                             cyclo(N-methyl)FYW.sub.D KV.Hcy.(CH.sub.2 CO.GGCRK.amide)                                              2.1 0.24                                                                              9                                           P587 cyclo(N-methyl)FYW.sub.D KV.Hcy.(CH.sub.2 CO.GGCK.amide) 3.4 0.61                                          6                                           P617 cyclo(N-methyl)FYW.sub.D KV.Hcy.(CH.sub.2 CO.GGCR.amide) 6.7 0.73                                          9                                           P726 cyclo(N-methyl)FYW.sub.D KV.Hcy.(CH.sub.2 CO.KKC.amide) 2.5 0.30       __________________________________________________________________________                                        8                                     

EXAMPLE 6

In Vivo Imaging of Human Somatostatin Receptor (SSTR)-Expressing Tumorswith Tc-99m Labeled P587

In a clinical trial, scintigraphic imaging of human patients bearingSSTR-expressing tumors was achieved using the Tc-99m labeled P587reagent.

A total of 10 patients, four females and six males ranging in age from27 to 69 years, had been previously diagnosed with growthhormone-secreting pituitary adenoma (4 patients), melanoma (1 subject),medullary thyroid cancer (1 subject), small cell lung carcinoma (SCLC; 1patient), non-Hodgkin's lymphoma (1 patient) or gastric carcinoid (1patient). Each of these patients were administered Tc-99m labeled P587at a dose of 10-22 mCi per 0.2-0.5 mg by intravenous injection.Scintigraphic imaging was them performed as described herein on eachpatient for 4 hours post-injection.

Gamma camera imaging is started simultaneously with injection. Anteriorimages were acquired as a dynamic study (10 sec image acquisitions) overthe first 10 min, and then as static images at 1, 2, 3 and 4 hpost-injection. Anterior images were acquired for 500,000 counts or 20min (whichever is shorter), at approximately 10-20 min, and atapproximately 1, 2, 3 and 4 h post-injection.

The scintigraphic imaging agent was found to clear rapidly from thebloodstream, resulting in less than 10% of the injected dose remainingin the circulation within 30 minutes of injection. This allowed imageacquisition of tumor sites to be achieved as early as 15-30 min afterinjection of scintigraphic imaging agent. All known tumors were detectedin this study, as well as two previously-undetected metastatic lesionswhich were later confirmed using computer-assisted tomography (CATscan).

These results demonstrated that the scintigraphic imaging agents of thisinvention were highly effective in detecting SSTR-expressing primary andmetastatic tumors in humans in vivo.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 1                                        - - <210> SEQ ID NO 1                                                        <211> LENGTH: 14                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: DISULFID                                                      <222> LOCATION: (3)..(14)                                                      - - <400> SEQUENCE: 1                                                         - -       Ala Gly Cys Lys Asn Phe Phe - #Trp Lys Thr Phe Thr Ser Cys                1          - #     5             - #     10                          __________________________________________________________________________

What is claimed is:
 1. A method for labeling a somatostatinreceptor-binding peptide having a formula

    cyclo(N--CH.sub.3)-Phe-Tyr-(D-Trp)-Lys-Val-Hcy.(CH.sub.2 CO.X)

wherein X is a metal ion complexing moiety having formula

    -(amino acid).sub.n -B-(amino acid).sub.m -Z,

wherein B is a thiol-containing moiety that is cysteine, homocysteine,isocysteine, or penicillamine; (amino acid)_(n) and (amino acid)_(m) areeach independently any primary α- or β-amino acid that does not comprisea thiol group; Z is --OH or --NH₂ ; n is an integer between 2 and 5; andm is an integer between 0 and 5;comprising the step of reacting thepeptide with technetium-99m in the presence of a reducing agent.
 2. Themethod of claim 1, wherein the reducing agent is a stannous ion.
 3. Amethod for labeling a somatostatin receptor-binding peptide having aformula:

    cyclo(N--CH.sub.3)-Phe-Tyr-(D-Trp)-Lys-Val-Hcy.(CH.sub.2 CO.GGCK.amide)

comprising the step of reacting the peptide with technetium-99m in thepresence of a reducing agent.
 4. The method of claim 3, wherein thereducing agent is a stannous ion.